The plant pathogen Botrytis cinerea (B. cinerea) causes severe plant diseases worldwide. Trichoderma is widely used as a biocontrol agent against B. cinerea through multiple biocontrol mechanisms. However, Trichoderma spp. with high biocontrol efficiency against B. cinerea under low-temperature conditions are barely reported. This study aimed to find potential low-temperature resistance biocontrol Trichoderma spp. against B. cinerea, and to characterize the biological principles underlying the activity of Trichoderma. Trichoderma koningiopsis (T. koningiopsis) C5-9 could fully overgrow a B. cinerea colony at 16 °C in a dual-culture assay. Treatment of cucumber leaves with T. koningiopsis C5-9 fermentation broth using the dipping method prior to B. cinerea inoculation significantly reduced the necrotic lesion diameter, with an inhibition rate of 55.30%. T. koningiopsis C5-9 could be successfully cultivated using the mycelia of B. cinerea as a carbon source at 16 °C. Transcriptomic analysis indicated that the origin recognition complex, organic substance catabolic process, and peroxisome were involved in the responses of T. koningiopsis C5-9 to B. cinerea. The findings of this study not only identified T. koningiopsis C5-9 as a potential biological control agent inhibiting B. cinerea under low-temperature conditions, but also provided new insights to develop a deeper understanding of the activity of Trichoderma against B. cinerea for plant protection.